Neuroblastoma (NB) is the most common extracranial solid tumor in pediatric patients. Treatment for high-risk patients includes surgery and high dose chemotherapy with autologous stem cell rescue. However, in spite of aggressive therapy, up to 80% of patients relapse and die of disseminated disease. Therefore, novel approaches to the treatment of NB are necessary. One potential approach is viral-directed enzyme prodrug therapy (VDEPT), using adenoviruses to deliver cDNAs encoding carboxylesterases (CEs) that efficiently activate the prodrug CPT-11. This drug has shown encouraging activity in NB patients, but is activated inefficiently in vivo. Work during the previous funding cycle documented that rabbit liver CE/CPT-11 VDEPT sensitizes primary NB cells to CPT-11, and can be used to purge NB cells from human hematopoietic "stem" cells without toxicity to progenitor cells or CD34+ NOD/SCID repopulating cells. In the next funding cycle, we propose to complete an ongoing nontherapeutic clinical trial for purging bone marrow specimens containing >1% tumor cells, and to identify appropriate CEs to make CE/CPT-11 VDEPT useful for in vivo application. Specific Aims include: 1) completion of the ongoing clinical trial; 2) modification of a human enzyme to produce an efficient CPT-11 activating enzyme with minimal immunogenicity; and 3) to achieve tumor-specific toxicity in mouse models of NB by using tumor specific promoters and replication-selective adenoviral vectors to deliver CE cDNA. Overall, these studies should provide alternative treatment modalities for high-risk neuroblastoma.